Lysine specific demethylase-1 (LSD1) serves as a regulator
of transcription
and represents a promising epigenetic target for anticancer treatment.
LSD1 inhibitors are in clinical trials for the treatment of Ewing’s
sarcoma (EWS), acute myeloid leukemia, and small cell lung cancer,
and the development of robust inhibitors requires accurate methods
for probing demethylation, potency, and selectivity. Here, the inhibition
kinetics on the H3K4me2 peptide and nucleosome substrates was examined,
comparing the rates of demethylation in the presence of reversible
[CC-90011 (PD) and SP-2577 (SD)] and irreversible [ORY-1001 (ID) and
tranylcypromine (TCP)] inhibitors. Inhibitors were also subject to
viability studies in three human cell lines and Western blot assays
to monitor H3K4me2 nucleosome levels in EWS (TC-32) cells, enabling
a correlation of drug potency, inhibition in vitro, and cell-based studies. For example, SP-2577, a drug in clinical
trials for EWS, inhibits activity on small peptide substrates (K
i = 60 ± 20 nM) using an indirect coupled
assay but does not inhibit demethylation on H3K4me2 peptides or nucleosomes
using direct Western blot approaches. In addition, the drug has no
effect on H3K4me2 levels in TC-32 cells. These data show that SP-2577
is not an LSD1 enzyme inhibitor, although the drug may function independent
of demethylation due to its cytotoxic selectivity in TC-32 cells.
Taken together, this work highlights the pitfalls of using coupled
assays to ascribe a drug’s mode of action, emphasizes the use
of physiologically relevant substrates in epigenetic drug targeting
strategies, and provides insight into the development of substrate-selective
inhibitors of LSD1.